A broad-host-range mobilizable shuttle vector for the construction of transcriptional fusions to β-galactosidase in Gram-positive bacteria
A broad-host-range mobilizable shuttle vector for the construction of transcriptional fusions to β-galactosidase in Gram-positive bacteria
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DOI：
10.1111/j.1574-6968.1997.tb12726.x
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发表时间：
1997-11-15
影响因子：
2.1
通讯作者：
Trieu-Cuot, P
中科院分区：
文献类型：
--
作者：
Poyart, C;Trieu-Cuot, P
构建了一种名为 pTCV-lac 的低拷贝数载体，为分析革兰氏阳性细菌中的调控元件提供了方便的系统。该载体的主要组成部分是：（i）pACYC184和广宿主范围肠球菌质粒pAM beta 1的复制起点，（ii）用于在革兰氏阴性和革兰氏阳性细菌中选择的红霉素和卡那霉素抗性编码基因，（iii）IncP质粒RK2的转移起点，以及（iv）无启动子的β-半乳糖苷酶编码lacZ基因，其具有革兰氏阳性核糖体结合位点。该 12 kb 质粒存在于革兰氏阳性宿主中，每个染色体当量有 3 至 5 个拷贝，并包含三个独特的克隆位点（EcoRI、SmaI、BamHI），用于克隆 lacZ 基因上游的 DNA 插入片段。携带不同启动子片段的质粒 pTCV-lac 和衍生物已通过接合从大肠杆菌 IncP 动员供体菌株转移至枯草芽孢杆菌、单核细胞增生李斯特氏菌、粪肠球菌和无乳链球菌。这些质粒在这些宿主中结构稳定，并且通过测定β-半乳糖苷酶比活性进行定量，发现相应的启动子活性覆盖了β-半乳糖苷酶值的至少100倍范围。这些结果表明 pTCV-lac 应该可用于分析多种革兰氏阳性细菌的基因调控。
A low-copy-number vector designated pTCV-lac has been constructed to provide a convenient system to analyze regulatory elements in Gram-positive bacteria. The main components of this vector are: (i) the origins of replication of pACYC184 and of the broad-host-range enterococcal plasmid pAM beta 1, (ii) erythromycin-and kanamycin-resistance-encoding genes for selection in Gram-negative and Gram-positive bacteria, (iii) the transfer origin of the IncP plasmid RK2, and (iv) a promoterless beta-galactosidase-encoding lacZ gene with a Gram-positive ribosome binding site. This 12 kb plasmid is present in Gram-positive hosts in three to five copies per chromosome equivalent and contains three unique cloning sites (EcoRI, SmaI, BamHI) for cloning of DNA inserts upstream of the lacZ gene. Plasmid pTCV-lac and derivatives carrying different promoter fragments have been transferred by conjugation from an Escherichia coli IncP mobilizing donor strain to Bacillus subtilis, Listeria monocytogenes, Enterococcus faecalis, and Streptococcus agalactiae. These plasmids were structurally stable in these hosts and the corresponding promoter activities, quantitated by the determination of the beta-galactosidase specific activities, were found to cover at least a 100-fold range in beta-galactosidase values. These results indicate that pTCV-lac should be useful for analysis of gene regulation in a wide range of Gram-positive bacteria.